Cefepime

Antimicrobial resistance profiling was performed using the most representative antimicrobial agents from the different antibiotic families, which are of great clinical and epidemiological relevance. Kirby-Bauer disk diffusion or broth microdilution methods (in the case of colistin) were done according to Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2020 ). Escherichia coli ATCC 25922 was used as a control strain. The antimicrobials tested by disk diffusion were: amoxicillin/clavulanate (20/10 μg), ampicillin (10 μg), aztreonam (30 μg), cefepime (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), fosfomycin (200 μg/50 μg of glucose-6-phosphate), gentamicin (10 μg), imipenem (10 μg), meropenem (10 μg), and trimethoprim-sulfamethoxazole (1.25/23.75 μg) (Becton Dickinson). Extended-spectrum beta-lactamases were screened by the ESBL test following the CLSI guidelines (CLSI, 2020 ). Isolates were classified as susceptible, resistant to 1 or 2 antimicrobial categories, multidrug-resistant (MDR) if resistant to at least one agent in ≥ 3 antimicrobial categories; or extensively drug-resistant (XDR), if resistant to at least one agent in all but two or fewer antimicrobial categories (Magiorakos et al., 2012 (link)).

Various β-lactamases in rods were screened using combination disk diffusion methods (Becton Dickinson, Sparks, MD, USA). ESBL enzymes were determined by DDTs (double-disk tests) using disks with cefotaxime (30 µg), ceftazidime (30 µg), cefepime (30 µg), and amoxicillin with clavulanic acid (30 µg) (Becton Dickinson, sparks, MD, USA). MBL was determined by a double-disk synergy test (DDST) using disks with imipenem (10 µg), ceftazidime (30 µg) (Becton Dickinson, sparks, MD, USA), and EDTA (standard). KPC enzymes were identified by a combined-disk test using disks of meropenem (10 µg) and meropenem (10 µg) with 10 µL of boronic acid (GRASO BIOTECH, Starogard Gdanski, Poland) [12 ,17 (link)]. The identification of the various types of carbapenemases was confirmed by the commercial test RESIN-4 O.K.N.V (Coris Bioconcept, Gembloux, Belgium). Immunochromatographic lateral flow assays were used for the rapid detection of OXA-48, KPC, NDM, and VIM carbapenemases from the cultured isolates (Argenta, Poznan, Poland) [18 (link)].
A quality control of all methods was performed using the following reference strains: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, Klebsiella pneumoniae ATCC 700603 (ESBL+), and Enterococcus faecalis ATCC 29212 and ATCC 51299 (VRE, HLGR).
All identifications were performed in triplicate [14 ].

The antimicrobial susceptibility testing was done following a previously reported method [26 (link)]. Briefly, the pure colonies obtained from blood agar were resuspended in bacterial suspensions, and were made depositing two or three medium-sized colonies (2 to 3 mm) in BBL Crystal Inoculum Broth (Becton Dickinson; Franklin Lakes, NJ, USA). The obtained inoculum was adjusted while using a Mac Farland 0.5 reading (Expected CFU/mL 1.5 × 108), and cultured in Müeller Hinton 150 × 15 mm² media BD BBL (Becton Dickinson Franklin Lakes, NJ, USA). The antibiotic discs were applied with the Sensi-Disc Designer Dispenser System. The antibiotics panel was conformed by ampicillin (10 µg), ampicillin/sulbactam (10/10 µg), mezlocillin (75 µg), carbenicillin (100 µg), piperacillin/tazobactam (100/10 µg), cefazolin (30 µg), cefaclor (30 µg), cefepime (30 µg), cefoperazone (75 µg), and cefotetan (30 µg) from Becton Dickinson (Franklin Lakes, NJ, USA), Müeller Hinton medium were incubated at 37 °C for 24 h in both aerobic and anaerobic conditions. In anaerobic conditions, the media were placed in an anaerobic jar (BD BBL™ GasPak™), with a C02 gas generators envelope (BD BBL™) and another envelope of BD BBL™ GasPak™ anaerobic indicator placed on them.